Method of preparing libraries of template polynucleotides

ABSTRACT

The present invention relates to a method for preparing a library of template polynucleotides and use thereof in methods of solid-phase nucleic acid amplification. More specifically, the invention relates to a method for preparing a library of template polynucleotides that have common sequences at their 5′ ends and at their 3′ ends.

FIELD OF THE INVENTION

The invention relates to a method of preparing a library of templatepolynucleotides and also the use of the library of templates in methodsof solid-phase nucleic acid amplification. In particular, the inventionrelates to a method of preparing a library of template polynucleotideswhich have common sequences at their 5′ ends and at their 3′ ends.

BACKGROUND TO THE INVENTION

Molecular biology and pharmaceutical drug development now make intensiveuse of nucleic acid analysis. The most challenging areas are wholegenome sequencing, single nucleotide polymorphism detection, screeningand gene expression monitoring, which typically require analysis oflarge amounts of nucleic acid.

One area of technology which revolutionised the study of nucleic acidswas the development of nucleic acid amplification techniques, such asthe polymerase chain reaction (PCR). Amplification reactions, such asPCR, can enable the user to specifically and selectively amplify aparticular target nucleic acid of interest from a complex mixture ofnucleic acids. However, there is also an ongoing need for nucleic acidamplification techniques which enable simultaneous amplification ofcomplex mixtures of templates of diverse sequence, such as genomic DNAfragments (e.g. “whole genome” amplification) or cDNA libraries, in asingle amplification reaction.

PCR amplification cannot occur in the absence of annealing of forwardand reverse amplification primers to primer binding sequences in thetemplate to be amplified under the conditions of the annealing steps ofthe PCR reaction, i.e. if there is insufficient complementarity betweenprimers and template. Some prior knowledge of the sequence of thetemplate is therefore required before one can carry out a PCR reactionto amplify a specific template, unless random primers are used with aconsequential loss of specificity. The user must usually know thesequence of at least the primer-binding sites in the template in advanceso that appropriate primers can be designed, although the remainingsequence of the template may be unknown. The need for prior knowledge ofthe sequence of the template increases the complexity and cost of PCRamplification of complex mixtures of templates, such as genomic DNAfragments.

WO 98/44151 and WO 00/18957 both describe methods of formingpolynucleotide arrays based on “solid-phase” nucleic acid amplification,which is analogous to a polymerase chain reaction wherein theamplification products are immobilised on a solid support in order toform arrays comprised of nucleic acid clusters or “colonies”. Eachcluster or colony on such an array is formed from a plurality ofidentical immobilised polynucleotide strands and a plurality ofidentical immobilised complementary polynucleotide strands. The arraysso-formed are generally referred to herein as “clustered arrays” andtheir general features will be further understood by reference to WO98/44151 or WO 00/18957, the contents of both documents beingincorporated herein in their entirety by reference.

As aforesaid, the solid-phase amplification methods of WO 98/44151 andWO 00/18957 are essentially a form of the polymerase chain reactioncarried out on a solid support. Like any PCR reaction these methodsrequire the use of forward and reverse amplification primers (which maybe identical or different) capable of annealing to a template to beamplified. In the methods of WO 98/44151 and WO 00/18957 both primersare immobilised on the solid support at the 5′ end. Other forms ofsolid-phase amplification are known in which only one primer isimmobilised and the other is present in free solution (Mitra, R. D andChurch, G. M., Nucleic Acids Research, 1999, Vol. 27, No. 24).

In common with all PCR techniques, solid-phase PCR amplificationrequires the use of forward and reverse amplification primers whichinclude “template-specific” nucleotide sequences which are capable ofannealing to sequences in the template to be amplified, or thecomplement thereof, under the conditions of the annealing steps of thePCR reaction. The sequences in the template to which the primers annealunder conditions of the PCR reaction may be referred to herein as“primer-binding” sequences.

Certain embodiments of the methods described in WO 98/44151 and WO00/18957 make use of “universal” primers to amplify templates comprisinga variable template portion that it is desired to amplify flanked 5′ and3′ by common or “universal” primer binding sequences. The “universal”forward and reverse primers include sequences capable of annealing tothe “universal” primer binding sequences in the template construct. Thevariable template portion may itself be of known, unknown or partiallyknown sequence. This approach has the advantage that it is not necessaryto design a specific pair of primers for each template to be amplified;the same primers can be used for amplification of different templatesprovided that each template is modified by addition of the sameuniversal primer-binding sequences to its 5′ and 3′ ends. The variabletemplate sequence can therefore be any DNA fragment of interest. Ananalogous approach can be used to amplify a mixture of templates, suchas a plurality or library of template nucleic acid molecules (e.g.genomic DNA fragments), using a single pair of universal forward andreverse primers, provided that each template molecule in the mixture ismodified by the addition of the same universal primer-binding sequences.

Such “universal primer” approaches to PCR amplification, and inparticular solid-phase PCR amplification, are advantageous since theyenable multiple template molecules of the same or different, known orunknown sequence to be amplified in a single amplification reaction,which may be carried out on a solid support bearing a single pair of“universal” primers. Simultaneous amplification of a mixture oftemplates of different sequences by PCR would otherwise require aplurality of primer pairs, each pair being complementary to each uniquetemplate in the mixture. The generation of a plurality of primer pairsfor each individual template is not a viable option for complex mixturesof templates.

The addition of universal priming sequences onto the ends of templatesto be amplified by PCR can be achieved by a variety of methods known tothose skilled in the art. For example, a universal primer consisting ofa universal sequence at its 5′ end and a degenerate sequence at its 3′end can be used in a PCR (DOP-PCR, eg PNAS 1996 vol 93 pg 14676-14679)to amplify fragments randomly from a complex template or a complexmixture of templates. The degenerate 3′ portion of the primer anneals atrandom positions on DNA and can be extended to generate a copy of thetemplate that has the universal sequence at its 5′ end.

Alternatively, adapters that contain universal priming sequences can beligated onto the ends of templates. The adapters may be single-strandedor double-stranded. If double-stranded, they may have overhanging endsthat are complementary to overhanging ends on the template moleculesthat have been generated with a restriction endonuclease. Alternatively,the double-stranded adapters may be blunt, in which case the templatesare also blunt ended. The blunt ends of the templates may have beenformed during a process to shear the DNA into fragments, or they mayhave been formed by an end repair reaction, as would be well known tothose skilled in the art.

A single adapter or two different adapters may be used in a ligationreaction with templates. If a template has been manipulated such thatits ends are the same, i.e. both are blunt or both have the sameoverhang, then ligation of a single compatible adapter will generate atemplate with that adapter on both ends. However, if two compatibleadapters, adapter A and adapter B, are used, then three permutations ofligated products are formed: template with adapter A on both ends,template with adapter B on both ends, and template with adapter A on oneend and adapter B on the other end. This last product is, under somecircumstances, the only desired product from the ligation reaction andconsequently additional purification steps are necessary following theligation reaction to purify it from the ligation products that have thesame adapter at both ends.

The current invention presented herein is a method that uses a singleadapter in a ligation reaction to generate a library of templatepolynucleotides each of which have common, but different, universalprimer sequences at their 5′ and 3′ ends. The method can be applied topreparing simple or complex mixes of templates for amplification, forexample a solid surface, using primer sequences, with no prior knowledgeof the template sequences. The invention is applicable to thepreparation of templates from complex samples such as whole genomes ormixtures of cDNAs, as well as mono-template applications.

SUMMARY OF THE INVENTION

In a first aspect the invention provides a method of generating alibrary of template polynucleotide molecules which have common sequencesat their 5′ ends and common sequences at their 3′ ends, the methodcomprising:

ligating identical mismatched adapter polynucleotides to both ends ofeach of one or more target polynucleotide duplexes to form one or moreadapter-target constructs, wherein each mismatched adapter is formedfrom two annealed polynucleotide strands that form a bimolecular complexcomprising at least one double-stranded region and an unmatched region,andcarrying out an initial primer extension reaction in which a primeroligonucleotide is annealed to an adapter portion of each of theadapter-target constructs and extended by sequential addition ofnucleotides to form extension products complementary to at least onestrand of each of the adapter-target constructs,wherein the extension products, and optionally amplification productsderived therefrom, collectively provide a library of templatepolynucleotide molecules which have common sequences at their 5′ endsand common sequences at their 3′ ends.

A second aspect of the invention relates to use of a library of templatepolynucleotide molecules prepared according to the method of the firstaspect of the invention as a template for solid-phase PCR amplification.Thus, in a particular embodiment the invention provides a method ofsolid-phase nucleic acid amplification of template polynucleotidemolecules which comprises:

preparing a library of template polynucleotide molecules which havecommon sequences at their 5′ and 3′ ends using the method according tothe first aspect of the invention and carrying out a solid-phase nucleicacid amplification reaction wherein said template polynucleotidemolecules are amplified.

A third aspect of the invention relates to use of a library of templatepolynucleotide molecules prepared according to the method of the firstaspect of the invention as a template for whole genome amplification.Thus, in a particular embodiment the invention provides a method ofwhole genome amplification which comprises:

using the method according to the first aspect of the invention toprepare a library of template polynucleotide molecules which have commonsequences at their 5′ and 3′ ends starting from a complex mixture ofwhole genome fragments and carrying out a whole genome amplificationreaction wherein said template polynucleotide molecules are amplified.

In a fourth aspect the invention provides a kit for use in preparing alibrary of template polynucleotide molecules which have common sequencesat their 5′ and 3′ ends wherein the common sequence at the 5′ end ofeach individual template in the library is not identical and not fullycomplementary to the common sequence at the 3′ end of said template, thekit comprising mismatched adapter polynucleotides as defined herein inrelation to the first aspect of the invention and oligonucleotideprimers capable of annealing to the mismatched adapter polynucleotides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates several examples of forked mismatched adapters foruse in the method of the invention, specifically depicting differentoverhanging or blunt end structures permissible at the “ligatable” endof the adapter. FIG. 1( e) schematically illustrates the sequencecomponents of the two partially complementary strands (denoted oligo Aand oligo B) which form the forked adapter when annealed. The 5′ end ofoligo B is complementary (COMP) to a part of the SEQ PRIMER sequence inoligo A. Oligo A includes a single “T” nucleotide overhang at the 3′end. The 5′ end of oligo A is phosphorylated. P represents a terminalphosphate group; X and Y represent surface capture functionalities.

FIG. 2 illustrates one embodiment of the method of the invention basedon use of the forked adapters illustrated in FIG. 1. FIG. 2( a) depictsthe steps of fragmenting a complex sample such as genomic DNA togenerate a plurality of target duplex fragments, ligation of the targetduplex fragments to mismatched (forked) adapters to generateadapter-template constructs and removal of unbound adapters. The forkedadapter includes a biotin group at the 5′ end, which is not ligated tothe target fragment, to facilitate solid-phase capture of theadapter-target constructs, e.g. onto streptavidin magnetic beads. FIG.2( b) depicts an initial primer extension reaction in which primers areannealed to mismatched adapter regions on each strand of anadapter-target construct and extended to generate extension productscomplementary to each strand of the adapter-target construct. Forsimplicity the ligation and primer extension steps are illustrated for asingle adapter-target construct.

FIG. 3 illustrates an alternative embodiment of the invention in whichadapter-target constructs are subjected to multiple rounds of primerannealing and extension to generate multiple single-stranded copies ofeach adapter-target construct. For simplicity the primer extension stepsare illustrated for a single adapter-target construct.

FIG. 4 illustrates a still further embodiment of the invention in whichadapter-target constructs are subjected to PCR amplification to generatemultiple double-stranded copies of each adapter-target construct. Forsimplicity PCR amplification illustrated for a single adapter-targetconstruct.

FIG. 5 illustrates an embodiment of the invention, depicting steps offragmenting a complex sample such as genomic DNA to generate a pluralityof target fragments, ligation of the target fragments to mismatched(forked) adapters to generate adapter-template constructs and subsequentremoval of unbound adapters, wherein the adapters do not include abiotin group at the 5′ end. The resulting adapter-target constructs maybe subjected to PCR amplification to generate multiple double-strandedcopies of each adapter-target construct. For simplicity the ligationsteps are illustrated for a single adapter-target construct.

FIG. 6 illustrates further examples of forked mismatched adapters foruse in the method of the invention, again depicting the permissibleblunt or overhang formats at the “ligatable” end of the adapter. FIG. 6(e) schematically illustrates the component sequences present in the twostrands (denoted oligo C and oligo B) which form the adapter whenannealed. P represents a terminal phosphate group; X and Y representsurface capture functionalities.

FIG. 7 illustrates a still further embodiment of the invention based onuse of the forked adapters illustrated in FIG. 6. FIG. 7( a) depictsfragmentation and ligation steps substantially similar to thoseillustrated in FIG. 5. FIG. 7( b) depicts subsequent PCR amplificationusing “tailed” PCR primers which have 3′ end sequences complementary toa sequence at the 5′ end of the adapter, and schematically illustratesthe sequence composition of the double-stranded amplification productsformed in the PCR reaction. For simplicity the ligation and PCRamplification steps are illustrated for a single adapter-targetconstruct.

FIG. 8 illustrates alternative embodiments of mismatched adapters foruse in the method of the invention. P represents a terminal phosphategroup; X and Y represent surface capture functionalities; X and Zrepresent modifications to prevent ligation.

FIG. 9 a illustrates a still further embodiment of the invention basedon use of the alternative adapters illustrated in FIG. 8. FIG. 9( a)depicts fragmentation, ligation and subsequent removal of unboundadapters. FIG. 9( b) depicts annealing of identical amplificationprimers to a double-stranded region of the adapter on each strand of theadapter-target construct. The adapter-target constructs can be amplifiedby PCR using this single primer species. For simplicity the ligationsteps and primer annealing are illustrated for a single adapter-targetconstruct.

DETAILED DESCRIPTION OF THE INVENTION

In its first aspect the invention provides a method of generating alibrary of template polynucleotide molecules which have common sequencesat their 5′ and 3′ ends. In this context the term “common” isinterpreted as meaning common to all templates in the library. Asexplained in further detail below, all templates within the library willcontain regions of common sequence at (or proximal to) their 5′ and 3′ends, wherein the common sequence at the 5′ end of each individualtemplate in the library is not identical and not fully complementary tothe common sequence at the 3′ end of said template.

The term “library” merely refers to a collection or plurality oftemplate molecules which share common sequences at their 5′ ends andcommon sequences at their 3′ ends. Use of the term “library” to refer toa collection or plurality of template molecules should not be taken toimply that the templates making up the library are derived from aparticular source, or that the “library” has a particular composition.By way of example, use of the term “library” should not be taken toimply that the individual templates within the library must be ofdifferent nucleotide sequence or that the templates be related in termsof sequence and/or source.

In it's various embodiments the invention encompasses formation ofso-called “monotemplate” libraries, which comprise multiple copies of asingle type of template molecule, each having common sequences at their5′ ends and their 3′ ends, as well as “complex” libraries wherein many,if not all, of the individual template molecules comprise differenttarget sequences (as defined below), although all share common sequencesat their 5′ ends and 3′ ends. Such complex template libraries may beprepared using the method of the invention starting from a complexmixture of target polynucleotides such as (but not limited to) randomgenomic DNA fragments, cDNA libraries etc. The invention also extends to“complex” libraries formed by mixing together several individual“monotemplate” libraries, each of which has been prepared separatelyusing the method of the invention starting from a single type of targetmolecule (i.e. a monotemplate). In preferred embodiments more than 50%,or more than 60%, or more than 70%, or more than 80%, or more than 90%,or more than 95% of the individual polynucleotide templates in a complexlibrary may comprise different target sequences, although all templatesin a given library will share common sequence at their 5′ ends andcommon sequence at their 3′ ends.

Use of the term “template” to refer to individual polynucleotidemolecules in the library merely indicates that one or both strands ofthe polynucleotides in the library are capable of acting as templatesfor template-dependent nucleic acid polymerisation catalysed by apolymerase. Use of this term should not be taken as limiting the scopeof the invention to libraries of polynucleotides which are actually usedas templates in a subsequent enzyme-catalysed polymerisation reaction.

The library is formed by ligating identical adapter polynucleotidemolecules (“mismatched adapters”, the general features of which aredefined below) to the 5′ and 3′ ends of one or more targetpolynucleotide duplexes (which may be of known, partially known orunknown sequence) to form adapter-target constructs and then carryingout an initial primer extension reaction in which extension productscomplementary to both strands of each individual adapter-targetconstruct are formed. The resulting primer extension products, andoptionally amplified copies thereof, collectively provide a library oftemplate polynucleotides.

Each strand of each template molecule in the library formed in theprimer extension reaction will therefore have the following structure,when viewed as a single strand:

5′-[common sequence I]-[target sequence]-[common sequence II]-3′wherein “common sequence I” represents a sequence derived from copying afirst strand of the mismatched adapter and is common to all templatemolecules in the library generated in the initial primer extensionreaction;“target” represents a sequence derived from one strand of the targetpolynucleotide duplex and may be different in different individualtemplate molecules within the library; and“common sequence II” represents a sequence derived from copying of asecond strand of the mismatched adapter and is also common to alltemplate molecules in the library generated in the initial primerextension reaction.

Since “common sequence I” and “common sequence II” are common to alltemplate strands in the library they may include “universal”primer-binding sequences, enabling all templates in the library to beultimately amplified in a solid-phase PCR procedure using universalprimers.

It is a key feature of the invention, however, that the common 5′ and 3′end sequences denoted “common sequence I” and “common sequence II” arenot fully complementary to each other, meaning that each individualtemplate strand can contain different (and non-complementary) universalprimer sequences at its 5′ and 3′ ends.

It is generally advantageous for complex libraries of templates to beamplified by PCR (e.g. whole genome amplification) whether performed insolution or on a solid support, to include regions of “different”sequence at their 5′ and 3′ ends, which are nevertheless common to alltemplate molecules in the library, especially if the amplificationproducts are to be ultimately sequenced. For example, the presence ofcommon unique sequence at one end only of each template in the librarycan provide a binding site for a sequencing primer, enabling one strandof each template in the amplified form of the library to be sequenced ina single sequencing reaction using a single type of sequencing primer.

Typically “common sequence I” and “common sequence II” will consist ofno more than 100, or no more than 50, or no more than 40 consecutivenucleotides at the 5′ and 3′ ends, respectively, of each strand of eachtemplate polynucleotide. The precise length of the two sequences may ormay not be identical. The nucleotide sequences of “common sequence I”and “common sequence II” in the template polynucleotides will bedetermined in part by the sequences of the adapter strands ligated tothe target polynucleotides and in part by the sequence of the primerused in the initial primer extension reaction, and any subsequent roundsof nucleic acid amplification.

In embodiments wherein the initial primer extension product is subjectedto further amplification by conventional PCR, then the products of theamplification reaction will be double-stranded polynucleotides, onestrand of which has the structure:

5′-[common sequence I]-[target sequence]-[common sequence II]-3′

It will be appreciated that “common sequence II” in the amplificationproducts may differ somewhat to the “common sequence II” present in theproducts of the initial primer extension reaction, since the former willbe determined in part by the sequence of the PCR primer used to primesynthesis of a polynucleotide strand complementary to the initial primerextension product, whereas the latter will be determined solely bycopying of the adapter sequences at the 3′ ends of the adapter-templateconstructs in the initial primer extension. Nevertheless, since the PCRprimer is designed to anneal to a sequence in the initial extensionproducts which is complementary to the 3′ adapter, the two forms of“common sequence II” will contain identical sequence, at least at the 3′end. Additional sequence may be included at the 5′ end of “commonsequence II” in the amplified products, for example by the use of“tailed” PCR primers, as described in detail below. In other embodimentsthe common sequences present in the amplification products may actuallybe shorter than the common sequences including in the adaptersoriginally ligated to the target.

The precise nucleotide sequences of the common regions of the templatemolecules in the library are generally not material to the invention andmay be selected by the user. The common sequences must at least comprise“primer-binding” sequences which enable specific annealing ofamplification primers when the templates are in use in a solid-phaseamplification reaction. The primer-binding sequences are thus determinedby the sequence of the primers to be ultimately used for solid-phaseamplification. The sequence of these primers in turn is advantageouslyselected to avoid or minimise binding of the primers to the targetportions of the templates within the library under the conditions of theamplification reaction, but is otherwise not particularly limited. Byway of example, if the target portions of the templates are derived fromhuman genomic DNA, then the sequences of the primers to be used in solidphase amplification should ideally be selected to minimise non-specificbinding to any human genomic sequence.

The adapter polynucleotides used in the method of the invention arereferred to herein as “mismatched” adapters because, as will beexplained in detail herein, it is essential that the adapters include aregion of sequence mismatch, i.e. they must not be formed by annealingof fully complementary polynucleotide strands.

Mismatched adapters for use in the invention are formed by annealing oftwo partially complementary polynucleotide strands so as to provide,when the two strands are annealed, at least one double-stranded regionand at least one unmatched region.

The “double-stranded region” of the adapter is a short double-strandedregion, typically comprising 5 or more consecutive base pairs, formed byannealing of the two partially complementary polynucleotide strands.This term simply refers to a double-stranded region of nucleic acid inwhich the two strands are annealed and does not imply any particularstructural conformation.

Generally it is advantageous for the double-stranded region to be asshort as possible without loss of function. By “function” in thiscontext is meant that the double-stranded region form a stable duplexunder standard reaction conditions for an enzyme-catalysed nucleic acidligation reaction, which will be well known to the skilled reader (e.g.incubation at a temperature in the range of from 4° C. to 25° C. in aligation buffer appropriate for the enzyme), such that the two strandsforming the adapter remain partially annealed during ligation of theadapter to a target molecule. It is not absolutely necessary for thedouble-stranded region to be stable under the conditions typically usedin the annealing steps of primer extension or PCR reactions.

Since identical adapters are ligated to both ends of each templatemolecule, the target sequence in each adapter-target construct will beflanked by complementary sequences derived from the double-strandedregion of the adapters.

The longer the double-stranded region, and hence the complementarysequences derived therefrom in the adapter-target constructs, thegreater the possibility that the adapter-target construct is able tofold back and base-pair to itself in these regions of internalself-complementarity under the annealing conditions used in primerextension and/or PCR. Generally it is preferred for the double-strandedregion to be 20 or less, 15 or less, or 10 or less base pairs in lengthin order to reduce this effect. The stability of the double-strandedregion may be increased, and hence its length potentially reduced, bythe inclusion of non-natural nucleotides which exhibit strongerbase-pairing than standard Watson-Crick base pairs.

It is preferred, but not absolutely essential, for the two strands ofthe adapter to be 100% complementary in the double-stranded region. Itwill be appreciated that one or more nucleotide mismatches may betolerated within the double-stranded region, provided that the twostrands are capable of forming a stable duplex under standard ligationconditions.

Adapters for use in the invention will generally include adouble-stranded region adjacent to the “ligatable” end of the adapter,i.e. the end that is joined to a target polynucleotide in the ligationreaction. The ligatable end of the adapter may be blunt or, in otherembodiments, short 5′ or 3′ overhangs of one or more nucleotides may bepresent to facilitate/promote ligation. The 5′ terminal nucleotide atthe ligatable end of the adapter should be phosphorylated to enablephosphodiester linkage to a 3′ hydroxyl group on the targetpolynucleotide.

The term “unmatched region” refers to a region of the adapter whereinthe sequences of the two polynucleotide strands forming the adapterexhibit a degree of non-complementarity such that the two strands arenot capable of annealing to each other under standard annealingconditions for a primer extension or PCR reaction. The two strands inthe unmatched region may exhibit some degree of annealing under standardreaction conditions for a enzyme-catalysed ligation reaction, providedthat the two strands revert to single stranded form under annealingconditions.

The conditions encountered during the annealing steps of a PCR reactionwill be generally known to one skilled in the art, although the preciseannealing conditions will vary from reaction to reaction (see Sambrooket al., 2001, Molecular Cloning, A Laboratory Manual, 3rd Ed, ColdSpring Harbor Laboratory Press, Cold Spring Harbor Laboratory Press, NY;Current Protocols, eds Ausubel et al.). Typically such conditions maycomprise, but are not limited to, (following a denaturing step at atemperature of about 94° C. for about one minute) exposure to atemperature in the range of from 40° C. to 72° C. (preferably 50-68° C.)for a period of about 1 minute in standard PCR reaction buffer.

Different annealing conditions may be used for a single primer extensionreaction not forming part of a PCR reaction (again see Sambrook et al.,2001, Molecular Cloning, A Laboratory Manual, 3rd Ed, Cold Spring HarborLaboratory Press, Cold Spring Harbor Laboratory Press, NY; CurrentProtocols, eds Ausubel et al.). Conditions for primer annealing in asingle primer extension include, for example, exposure to a temperaturein the range of from 30 to 37° C. in standard primer extension buffer.It will be appreciated that different enzymes, and hence differentreaction buffers, may be used for a single primer extension reaction asopposed to a PCR reaction. There is no requirement to use a thermostablepolymerase for a single primer extension reaction.

It is to be understood that the “unmatched region” is provided bydifferent portions of the same two polynucleotide strands which form thedouble-stranded region(s). However, the portions of the two strandsforming the unmatched region are not annealed under conditions in whichother portions of the same two strands are annealed to form one or moredouble-stranded regions. For avoidance of doubt it is to be understoodthat a single-stranded or single base overhang at the 5′ or 3′ end of apolynucleotide duplex does not constitute an “unmatched region” in thecontext of this invention.

The portions of the two strands forming the double-stranded regiontypically comprise at least 10, or at least 15, or at least 20consecutive nucleotides on each strand. The lower limit on the length ofthe unmatched region will typically be determined by function, forexample the need to provide a suitable sequence for binding of a primerfor primer extension, PCR and/or sequencing. Theoretically there is noupper limit on the length of the unmatched region, except that itgeneral it is advantageous to minimise the overall length of theadapter, for example in order to facilitate separation of unboundadapters from adapter-target constructs following the ligation step.Therefore, it is preferred that the unmatched region should be less than50, or less than 40, or less than 30, or less than 25 consecutivenucleotides in length on each strand.

The overall length of the two strands forming the adapter will typicallyin the range of from 25 to 100 nucleotides, more typically from 30 to 55nucleotides.

The portions of the two strands forming the unmatched region shouldpreferably be of similar length, although this is not absolutelyessential, provided that the length of each portion is sufficient tofulfil its desired function (e.g. primer binding). The inventors' haveshown by experiment that the portions of the two strands forming theunmatched region may differ by up to 25 nucleotides without undulyaffecting adapter function.

Most preferably the portions of the two polynucleotide strands formingthe unmatched region will be completely mismatched, or 100%non-complementary. However, skilled readers will be appreciate that somesequence “matches”, i.e. a lesser degree of non-complementarity may betolerated in this region without affecting function to a materialextent. As aforesaid, the extent of sequence mismatching ornon-complementarity must be such that the two strands in the unmatchedregion remain in single-stranded form under annealing conditions asdefined above.

The precise nucleotide sequence of the adapters is generally notmaterial to the invention and may be selected by the user such that thedesired sequence elements are ultimately included in the commonsequences of the library of templates derived from the adapters, forexample to provide binding sites for particular sets of universalamplification primers and/or sequencing primers. Additional sequenceelements may be included, for example to provide binding sites forsequencing primers which will ultimately be used in sequencing oftemplate molecules in the library, or products derived fromamplification of the template library, for example on a solid support.The adapters may further include “tag” sequences, which can be used totag or mark template molecules derived from a particular source.

The general features and use of such tag sequences is described in theapplicant's pending application published as WO 05/068656.

Although the precise nucleotide sequence of the adapter is generallynon-limiting to the invention, the sequences of the individual strandsin the unmatched region should be such that neither individual strandexhibits any internal self-complementarity which could lead toself-annealing, formation of hairpin structures etc. under standardannealing conditions. Self-annealing of a strand in the unmatched regionis to be avoided as it may prevent or reduce specific binding of anamplification primer to this strand.

The mismatched adapters are preferably formed from two strands of DNA,but may include mixtures of natural and non-natural nucleotides (e.g.one or more ribonucleotides) linked by a mixture of phosphodiester andnon-phosphodiester backbone linkages. Other non-nucleotide modificationsmay be included such as, for example, biotin moieties, blocking groupsand capture moieties for attachment to a solid surface, as discussed infurther detail below.

The one or more “target polynucleotide duplexes” to which the adaptersare ligated may be any polynucleotide molecules that it is desired toamplify by solid-phase PCR, generally with a view to sequencing. Thetarget polynucleotide duplexes may originate in double-stranded DNA form(e.g. genomic DNA fragments) or may have originated in single-strandedform, as DNA or RNA, and been converted to dsDNA form prior to ligation.By way of example, mRNA molecules may be copied into double-strandedcDNAs suitable for use in the method of the invention using standardtechniques well known in the art. The precise sequence of the targetmolecules is generally not material to the invention, and may be knownor unknown. Modified DNA molecules including non-natural nucleotidesand/or non-natural backbone linkages could serve as the target, providedthat the modifications do not preclude adapter ligation and/or copyingin a primer extension reaction.

Although the method could in theory be applied to a single target duplex(i.e. one individual double-stranded molecule), it is preferred to use amixture or plurality of target polynucleotide duplexes. The method ofthe invention may be applied to multiple copies of the same targetmolecule (so-called monotemplate applications) or to a mixture ofdifferent target molecules which differ from each other with respect tonucleotide sequence over all or a part of their length, e.g. a complexmixture of templates. The method may be applied to a plurality of targetmolecules derived from a common source, for example a library of genomicDNA fragments derived from a particular individual. In a preferredembodiment the target polynucleotides will comprise random fragments ofhuman genomic DNA. The fragments may be derived from a whole genome orfrom part of a genome (e.g. a single chromosome or sub-fractionthereof), and from one individual or several individuals. The DNA targetmolecules may be treated chemically or enzymatically either prior to, orsubsequent to the ligation of the adaptor sequences. Techniques forfragmentation of genomic DNA include, for example, enzymatic digestionor mechanical shearing.

“Ligation” of adapters to 5′ and 3′ ends of each target polynucleotideinvolves joining of the two polynucleotide strands of the adapter todouble-stranded target polynucleotide such that covalent linkages areformed between both strands of the two double-stranded molecules. Inthis context “joining” means covalent linkage of two polynucleotidestrands which were not previously covalently linked. Preferably such“joining” will take place by formation of a phosphodiester linkagebetween the two polynucleotide strands but other means of covalentlinkage (e.g. non-phosphodiester backbone linkages) may be used.However, it is an essential requirement that the covalent linkagesformed in the ligation reactions allow for read-through of a polymerase,such that the resultant construct can be copied in a primer extensionreaction using primers which binding to sequences in the regions of theadapter-target construct that are derived from the adapter molecules.

The ligation reactions will preferably be enzyme-catalysed. The natureof the ligase enzyme used for enzymatic ligation is not particularlylimited. Non-enzymatic ligation techniques (e.g. chemical ligation) mayalso be used, provided that the non-enzymatic ligation leads to theformation of a covalent linkage which allows read-through of apolymerase, such that the resultant construct can be copied in a primerextension reaction.

The desired products of the ligation reaction are adapter-targetconstructs in which identical adapters are ligated at both ends of eachtarget polynucleotide, given the structure adapter-target-adapter.Conditions of the ligation reaction should therefore be optimised tomaximise the formation of this product, in preference to targets havingan adapter at one end only.

The products of the ligation reaction may be subjected to purificationsteps in order to remove unbound adapter molecules before theadapter-target constructs are processed further. Any suitable techniquemay be used to remove excess unbound adapters, preferred examples ofwhich will be described in further detail below.

Adapter-target constructs formed in the ligation reaction are thensubject to an initial primer extension reaction in which a primeroligonucleotide is annealed to an adapter portion of each of theadapter-target constructs and extended by sequential addition ofnucleotides to the free 3′ hydroxyl end of the primer to form extensionproducts complementary to at least one strand of each of theadapter-target constructs.

The term “initial” primer extension reaction refers to a primerextension reaction in which primers are annealed directly to theadapter-target constructs, as opposed to either complementary strandsformed by primer extension using the adapter-target construct as atemplate or amplified copies of the adapter-target construct. It is akey feature of the method of the invention that the initial primerextension reaction is carried out using a “universal” primer which bindsspecifically to a cognate sequence within an adapter portion of theadapter-target construct, and is not carried out using a target-specificprimer or a mixture of random primers. The use of an adapter-specificprimer for the initial primer extension reaction is key to formation ofa library of templates which have common sequence at the 5′ and commonsequence at the 3′ end.

The primers used for the initial primer extension reaction will becapable of annealing to each individual strand of adapter-targetconstructs having adapters ligated at both ends, and can be extended soas to obtain two separate primer extension products, one complementaryto each strand of the construct. Thus, in the most preferred embodimentthe initial primer extension reaction will result in formation of primerextension products complementary to each strand of each adapter-target

In a preferred embodiment the primer used in the initial primerextension reaction will anneal to a primer-binding sequence (in onestrand) in the unmatched region of the adapter.

The term “annealing” as used in this context refers to sequence-specificbinding/hybridisation of the primer to a primer-binding sequence in anadapter region of the adapter-target construct under the conditions tobe used for the primer annealing step of the initial primer extensionreaction.

The products of the primer extension reaction may be subjected tostandard denaturing conditions in order to separate the extensionproducts from strands of the adapter-target constructs. Optionally thestrands of the adapter-target constructs may be removed at this stage.The extension products (with or without the original strands of theadapter-target constructs) collectively form a library of templatepolynucleotides which can be used as templates for solid-phase PCR.

If desired, the initial primer extension reaction may be repeated one ormore times, through rounds of primer annealing, extension anddenaturation, in order to form multiple copies of the same extensionproducts complementary to the adapter-target constructs.

In other embodiments the initial extension products may be amplified byconvention solution-phase PCR, as described in further detail below. Theproducts of such further PCR amplification may be collected to form alibrary of templates comprising “amplification products derived from”the initial primer extension products. In a preferred embodiment bothprimers used for further PCR amplification will anneal to differentprimer-binding sequences on opposite strands in the unmatched region ofthe adapter. Other embodiments may, however, be based on the use of asingle type of amplification primer which anneals to a primer-bindingsequence in the double-stranded region of the adapter. In embodiments ofthe method based on PCR amplification the “initial” primer extensionreaction occurs in the first cycle of PCR.

Inclusion of the initial primer extension step (and optionally furtherrounds of PCR amplification) to form complementary copies of theadapter-target constructs (prior to whole genome or solid-phase PCR) isadvantageous, for several reasons. Firstly, inclusion of the primerextension step, and subsequent PCR amplification, acts as an enrichmentstep to select for adapter-target constructs with adapters ligated atboth ends. Only target constructs with adapters ligated at both endsprovide effective templates for whole genome or solid-phase PCR usingcommon or universal primers specific for primer-binding sequences in theadapters, hence it is advantageous to produce a template librarycomprising only double-ligated targets prior to solid-phase or wholegenome amplification.

Secondly, inclusion of the initial primer extension step, and subsequentPCR amplification, permits the length of the common sequences at the 5′and 3′ ends of the target to be increased prior to solid-phase or wholegenome PCR. As outlined above, it is generally advantageous for thelength of the adapter molecules to be kept as short as possible, tomaximise the efficiency of ligation and subsequent removal of unboundadapters. However, for the purposes of whole genome or solid-phase PCRit may be an advantage to have longer sequences common or “universal”sequences at the 5′ and 3′ ends of the templates to be amplified.Inclusion of the primer extension (and subsequent amplification) stepsmeans that the length of the common sequences at one (or both) ends ofthe polynucleotides in the template library can be increased afterligation by inclusion of additional sequence at the 5′ ends of theprimers used for primer extension (and subsequent amplification). Theuse of such “tailed” primers is described in further detail below.

Various non-limiting specific embodiments of the method of the inventionwill now be described in further detail with reference to theaccompanying drawings. Features described as being preferred in relationto one specific embodiment of the invention apply mutatis mutandis toother specific embodiments of the invention unless stated otherwise.

FIG. 1 illustrates several embodiments of a particular type ofmismatched adapter for use in the method of the invention. The adapteris formed by annealing two single-stranded oligonucleotides, hereinreferred to as “oligo A” and “oligo B”. Oligo A and oligo B may beprepared by conventional automated oligonucleotide synthesis techniquesin routine use in the art. The oligonucleotides are partiallycomplementary such that the 3′ end of oligo A is complementary to the 5′end of oligo B. The 5′ end of oligo A and the 3′ end of oligo B are notcomplementary to each other. When the two strands are annealed, theresulting structure is double stranded at one end (the double-strandedregion) and single stranded at the other end (the unmatched region) andis referred to herein as a “forked adapter” (FIG. 1 a). Thedouble-stranded region of the forked adapter may be blunt-ended (FIG. 1b) or it may have an overhang. In the latter case, the overhang may be a3′ overhang (FIG. 1 c) or a 5′ overhang (FIG. 1 d), and may comprise asingle nucleotide or more than one nucleotide.

The 5′ end of the double-stranded part of the forked adapter isphosphorylated, i.e. the 5′ end of oligo B (FIG. 1 a-d). The presence ofthe 5′ phosphosphate group identifies this as the “ligatable” end of theadapter. The 5′ end of oligo A may be biotinylated or bear anotherfunctionality (represented by X) that enables it to be captured on asurface, such as a bead. Such alternative functionalities other thanbiotin are known to those skilled in the art. The 3′ end of oligo B mayalso be biotinylated or bear another functionality (represented by Y)that enables it to be captured on a surface (FIG. 1 d).

The phosphodiester bonds that comprise the back-bone of theoligonucleotides may be replaced with non-enzymatically cleavable bondssuch as phosphorothioate bonds. Preferably only the last, or last andpenultimate, phosphodiester bonds at both the 3′ and 5′ ends of theoligonucleotides will be substituted with phosphorothioate bonds. In themost preferred embodiment of the invention, oligo A contains a biotingroup on its 5′ end, oligo B is phosphorylated at its 5′ end and thedouble-stranded portion of the duplex contains a single base 3′ overhangcomprising a ‘T’ nucleotide. Oligo A consists of two sequences: asequence at the 5′ end which is identical to that of a universal primerto be used for PCR amplification, referred to herein as “PRIMER 1”sequence, and at its 3′ end a sequence identical to that of a universalsequencing primer, herein referred to herein as “SEQ PRIMER” sequence,plus an additional ‘T’ nucleotide on the 3′ end. Oligo B also consistsof two sequences: a sequence at its 5′ end that is complementary to onlypart of the 3′ end of the SEQ PRIMER sequence in Oligo A, excluding the‘T’ overhang of Oligo A, and a sequence complementary to that of auniversal PCR amplification primer, herein referred to as “comp-PRIMER2” at its 3′ end (FIG. 1 e).

FIG. 2 illustrates one embodiment of the method of the invention basedon use of the forked adapters illustrated in FIG. 1. A mixture of targetDNA molecules of different sequence may be prepared by mixing a number,greater than one, of individual DNA molecules. In the preferredprocedure, genomic DNA is fragmented into small molecules, preferablyless than 1000 base pairs in length. Fragmentation of DNA may beachieved by a number of methods including: enzymatic digestion, chemicalcleavage, sonication, nebulisation, or hydroshearing, preferablynebulisation.

Fragmented DNA may be made blunt-ended by a number of methods known tothose skilled in the art. In the preferred method, the ends of thefragmented DNA are end repaired with T4 DNA polymerase and Klenowpolymerase, a procedure well known to those skilled in the art, and thenphosphorylated with a polynucleotide kinase enzyme. A single ‘A’deoxynucleotide is then added to both 3′ ends of the DNA molecules usingTaq polymerase enzyme, producing a one-base 3′ overhang that iscomplementary to the one-base 3″T′ overhang on the double-stranded endof the forked adapter.

A ligation reaction between the forked adapter and the DNA fragments isthen performed using a suitable ligase enzyme (e.g. T4 DNA ligase) whichjoins two copies of the adapter to each DNA fragment, one at either end,to form adapter-target constructs. The products of this reaction can bepurified from unligated adapter by a number of means, includingsize-inclusion chromatography, preferably by electrophoresis through anagarose gel slab followed by excision of a portion of the agarose thatcontains the DNA greater in size that the size of the adapter (FIG. 2a).

After the excess adapter has been removed, unligated target DNA remainsin addition to ligated adapter-target constructs and this can be removedby selectively capturing only those target DNA molecules that haveadapter attached. The presence of a biotin group on the 5′ end of OligoA of the adapter enables any target DNA ligated to the adapter to becaptured on a surface coated with streptavidin, a protein thatselectively and tightly binds biotin. Streptavidin can be coated onto asurface by means known to those skilled in the art. In the preferredmethod, commercially available magnetic beads that are coated instreptavidin can be used to capture ligated adapter-target constructs.The application of a magnet to the side of a tube containing these beadsimmobilises them such that they can be washed free of the unligatedtarget DNA molecules (FIG. 2 a).

An oligonucleotide, herein referred to as PRIMER 2, which hybridises tothe “comp-PRIMER 2” sequence on the oligo B strand of the adapter-targetconstructs can be used in an initial primer extension reaction togenerate a complementary copy of the adapter-target strand attached tothe bead. The resulting primer extension product forms a double-strandedduplex with its complementary adapter-target strand attached to the beadand it can then be isolated and purified from adapter-target strand onthe bead by denaturation (FIG. 2 b).

There are several standard methods for separating the strand of a DNAduplex by denaturation, including thermal denaturation, or preferablychemical denaturation in 100 mM sodium hydroxide solution. The pH of asolution of single-stranded DNA in a sodium hydroxide collected from thesupernatant of a suspension of magnetic beads can be neutralised byadjusting with an appropriate solution of acid, or preferably bybuffer-exchange through a size-exclusion chromatography columnpre-equilibrated in a buffered solution. The resulting solution containsa library of single-stranded DNA template molecules all of whichcomprise in order: 5′ PRIMER 2 sequence, target DNA, the complement ofSEQ PRIMER sequence, then the complement of PRIMER 1 sequence. Thistemplate library can then be used for solid-phase PCR amplificationusing immobilised PRIMER 1 and PRIMER 2 oligonucleotides. It will beappreciated, however, that the utility of the library is not limited tosolid-phase PCR but extends to any type of PCR amplification, includingwhole-genome amplification performed entirely in solution.

FIG. 3 illustrates an alternative embodiment of the invention in whichadapter-target constructs prepared as described above with reference toFIG. 2 are subjected to multiple rounds of primer annealing andextension to generate multiple single-stranded copies of eachadapter-target construct. In this embodiment of the invention, theinitial primer extension reaction on the bead-immobilisedadapter-template molecules with PRIMER 2 is in effect replaced with anasymmetric PCR amplification with the PRIMER 2 oligonucleotide (FIG. 3),this being equivalent to multiple rounds of the same primer extensionreaction. In this embodiment, multiple single-stranded copies of thebead-immobilised strands are generated in the supernatant of the beadsuspension due to PCR thermocycling, hence a separate denaturation stepis not necessary to recover the newly synthesised complementary copiesof the bead-immobilised adapter-target strands; the copies can bepurified from the supernatant by standard methods, known to thoseskilled in the art.

In another embodiment of the invention, illustrated in FIG. 4, theinitial primer extension reaction on the bead-immobilised adapter-targetconstructs with PRIMER 2 forms part of a standard (symmetric) PCRamplification with the PRIMER 2 and PRIMER 1 oligonucleotides. In thisembodiment, multiple double-stranded copies of the bead-immobilisedstrands are generated in the supernatant of the bead suspension due toPCR thermocycling, hence a separate denaturation step is not necessaryto recover the newly synthesised complementary copies of thebead-immobilised adapter-target strands; the copies can be purified fromthe supernatant by standard methods, known to those skilled in the art.

In another embodiment of the invention, illustrated in FIG. 5, theforked adapter does not contain a biotin group at the 5′ end of theOligo A strand. In this embodiment, fragmented DNA may be madeblunt-ended by a number of methods know to those skilled in the art. Inthe preferred method, the ends of the fragmented are end repaired withT4 DNA polymerase and Klenow polymerase, and then phosphorylated withpolynucleotide kinase enzyme. A single ‘A’ deoxynucleotide is then addedto both 3′ ends of the DNA molecules with Taq polymerase enzyme,producing a one-base 3′ overhang that is complementary to the one-base3″T′ overhang on the double-stranded “ligatable” end of the forkedadapter. A ligation reaction between the forked adapter and the DNAfragments is then performed, e.g. using T4 DNA ligase enzyme, whichjoins two copies of the adapter to each DNA template molecule, one ateither end.

The products of the ligation reaction can be purified from unligatedadapter by a number of means, including size-inclusion chromatography,preferably by electrophoresis through an agarose gel slab followed byexcision of a portion of the agarose that contains the DNA greater insize that the size of the adapter. An aliquot of the purified DNA isthen used in a PCR amplification with the PRIMER 2 and PRIMER 1oligonucleotides (FIG. 5). The first PCR cycle will involve an initialprimer extension reaction with primer 2 (not illustrated). The primersselectively amplify those template DNA molecules that have adaptersligated on both ends. The product of the reaction is a library ofdouble-stranded template molecules, each of which comprise in order onone of the duplex strands: 5′ PRIMER 2 sequence, target DNA, thecomplement of SEQ PRIMER sequence, then the complement of PRIMER 1sequence. This library can then be used on a solid-phase PCR platformthat contains immobilised PRIMER 1 and PRIMER 2 oligonucleotides.

FIG. 6 illustrates further examples of forked mismatched adapters foruse in the method of the invention. In this embodiment the forkedadapter is formed by annealing two single-stranded oligonucleotides,herein referred to as “oligo C” and “oligo B”. The oligonucleotides arepartially complementary such that the 3′ end of oligo C is complementaryto the 5′ end of oligo B. The 5′ end of oligo C and the 3′ end of oligoB are not complementary to each other. When the two oligos are annealedthe resulting structure is double-stranded at one end (double-strandedregion) and single-stranded at the other end (unmatched region) (FIG. 6a). The double-stranded region of the forked adapter may be blunt-ended(FIG. 6 d) or it may have an overhang. In the latter case, the overhangmay be a 3′ overhang (FIG. 6 c) or a 5′ overhang (FIG. 6 b), and maycomprise a single base or more than one base.

The 5′ end of the double-stranded region of the forked adapter isphosphorylated i.e. the 5′ end of ‘oligo B’(FIG. 6 a-d) to provide a“ligatable” end. The 5′ end of oligo C may be biotinylated or bearanother functionality (X) that enables it to be captured on a surface,such as a bead. The 3′ end of oligo B may also be biotinylated or bearanother functionality (Y) that enables it to be captured on a surface(FIG. 6 d).

The phosphodiester bonds that comprise the back-bone of theoligonucleotides may be replaced with non-enzymatically cleavable bondssuch as phosphorothioate bonds. Preferably only the last, or last andpenultimate, phosphodiester bonds at both the 3′ and 5′ ends of theoligonucleotides will be substituted with phosphorothioate bonds. OligoC consists of only one sequence: a sequence identical to that of auniversal sequencing primer denoted “SEQ PRIMER” (or identical to partof the 3′ end of the “SEQ PRIMER” sequence), plus an additional ‘T’nucleotide on the 3′ end. Oligo B consists of two sequences: a sequenceat its 5′ end that is complementary to only part of the 3′ end of theSEQ PRIMER sequence in Oligo C, excluding the ‘T’ overhang of ‘Oligo C’,and a sequence at its 3′ end which is complementary to that of auniversal PCR amplification primer, herein referred to as the“comp-PRIMER 2” sequence, (FIG. 6 e).

FIG. 7 illustrates a further embodiment of the invention based on use ofthe forked adapters illustrated in FIG. 6. In this embodiment,adapter-target constructs are prepared substantially as described abovewith reference to FIG. 5, except that the adapters illustrated in FIG. 6are used (FIG. 7 a).

An aliquot of the purified adapter-target constructs is used in astandard solution-phase PCR amplification with “tailed” primeroligonucleotides. Tailed primers are primers that only hybridize viatheir 3′ end to a target sequence, leaving a 5′ non-hybridised tail. Thelength and precise sequence of the non-hybridised tail is non-limiting,but may typically comprise from 10 to 50 nucleotides, more typicallyfrom 15 to 30 nucleotides. When used in amplifications by PCR, theinitial round of PCR amplification (i.e. the first and second primerextension reactions) rely on binding of the 3′ ends of the tailedprimers to cognate primer-binding sequences in the adapter regions ofthe adapter-target constructs. The 5′ non-hybridising tails derived fromthe tailed primers act as templates in subsequent PCR cycles and aretherefore copied into the resultant double-stranded PCR products.

In the current embodiment, either one or both of the primers used in theamplification reaction can be “tailed” primers. In one embodiment, theprimers used are denoted PRIMER 3 and PRIMER 4, where PRIMER 3 consistsof a 5′ tail sequence, and a 3′ sequence that is complementary to the“comp PRIMER 2” sequence in the forked adapter; and PRIMER 4 consists ofa 5′ tail sequence, and a 3′ sequence that is identical to the 5′ end ofthe SEQ PRIMER sequence present in the unmatched region of the forkedadapter. Following amplification by PCR, the tail sequences areincorporated into the copies of the adapter-target DNA construct.

In one embodiment of the invention, the tail sequences on PRIMER 3 andPRIMER 4 are non-identical sequences. The tail sequence of PRIMER 3 andthe tail sequence of PRIMER 4 can then be used to form the sequence ofsurface-immobilised primers to be used on a solid-phase DNAamplification platform (FIG. 7 b).

In another embodiment of the invention, the tail sequences on PRIMER 3and PRIMER 4 are identical sequences. The products of the solution-phasePCR will thus have the same sequence at their ends: the common tailsequence of PRIMER 3 and PRIMER 4. This common tail sequence can then beused to form the sequence of a single surface-immobilised primer on asolid-phase DNA amplification platform. Surface amplification of thelibrary of templates may thus be performed using a single PCR primerimmobilised on the surface.

FIG. 8 illustrates alternative embodiments of mismatched adapters foruse in the method of the invention. These “modified” forked adapters maybe designed to enable solid-phase amplification of templates using asingle surface bound primer. The adapter is formed by annealing twosingle-stranded oligonucleotides, herein referred to as “oligo D” and“oligo E”. The oligonucleotides are partially complementary such thatthe 3′ end of oligo D is complementary to the 5′ end of oligo E, and the5′ end of oligo D is complementary to the 3′ end of oligo E, however thecentral portions of oligo D and oligo E are non-complementary. When theoligo D and oligo E are annealed the resulting structure is doublestranded at both ends (double-stranded regions) and single stranded inthe middle (unmatched region) and is referred to herein as the “modifiedForked adapter” (FIG. 8 a).

One end of the modified forked adapter is modified to prevent ligationof a DNA molecule to this end. Such modifications are known to thoseskilled in the art and may include, for example, the presence of a 5′ or3′ overhang. The other “ligatable” end may be blunt-ended (FIG. 8 d) ormay have an overhang. In the latter case, the overhang may be a 3′overhang (FIG. 8 c) or a 5′ overhang (FIG. 8 b), and may comprise asingle base or more than one base. The 5′ strand of the ligatable end isphosphorylated i.e. the 5′ end of oligo E (FIG. 8 a-d). The 5′ end ofoligo D may be biotinylated or bear another functionality that enablesit to be captured on a surface, such as a bead. The 3′ end of oligo Emay be biotinylated or bear another functionality that enables it to becaptured on a surface (FIG. 8 d). The modifications to prevent ligation(Z,W) may be the same as or different to the surface capturefunctionalities (X,Y).

The phosphodiester bonds that comprise the back-bone of theoligonucleotides may be replaced with non-enzymatically cleavable bondssuch as phosphorothioate bonds. Preferably only the last, or last andpenultimate, phosphodiester bonds at both the 3′ and 5′ ends of theoligonucleotides will be substituted with phosphorothioate bonds.

In the preferred embodiment of the invention, oligo E is phosphorylatedat its 5′ end and the 3′ end of oligo D contains a single base 3′overhang comprising a “T” nucleotide. Oligo D consists of two sequences:a sequence at its 5′ end which is identical to that of a universal PCRamplification primer, referred to herein as “PRIMER 5” sequence, next toa sequence identical to that of a universal sequencing primer denoted“SEQ PRIMER” sequence plus the additional “T” nucleotide on the 3′ end.Oligo E consists of three sequences: a sequence at its 5′ end that iscomplementary to only part of the 3′ end of the SEQ PRIMER sequence inOligo D, excluding the ‘T’ overhang of Oligo D, a central sequencenon-complementary to any part of Oligo D, and a 3′ end that iscomplementary to the “PRIMER 5” sequence of Oligo D (FIG. 8 e).

FIG. 9 illustrates a still further embodiment of the invention based onuse of the alternative adapters illustrated in FIG. 8. In thisembodiment adapter-target constructs may be prepared substantially asdescribed above in relation to FIG. 5, except that the modified forkedadapters illustrated in FIG. 8 are used. An aliquot of the purifiedadapter-target constructs is used in a solution-phase PCR amplificationusing PRIMER 5 oligonucleotide to selectively amplify those ligationproducts that have the modified adapter on both ends (FIG. 9 b). Theproduct of the solution-phase PCR can then be purified and amplified ona solid-phase PCR platform with a single immobilised primer, e.g. PRIMER5. Inclusion of the mismatch sequence in oligo E ensures that allproducts of this solid-phase amplification will contain commonsequencing primer binding sequences on one strand only, enablingsequencing using a universal sequencing primer which anneals to thiscommon sequence.

Use of the Template Library

Template libraries prepared according to the method of the invention maybe used in essentially any method of nucleic acid analysis whichrequires further amplification of the templates and/or sequencing of thetemplates or amplification products thereof. Exemplary uses of thetemplate libraries include, but are not limited to, providing templatesfor whole genome amplification and also solid-phase PCR amplification(of either monotemplate or complex template libraries). A particularlypreferred use is in whole-genome amplification carried out on asolid-support.

Whole-Genome Amplification

Template libraries prepared according to the method of the inventionstarting from a complex mixture of genomic DNA fragments representing awhole or substantially whole genome provide suitable templates forso-called “whole-genome” amplification. The term “whole-genomeamplification” refers to a nucleic acid amplification reaction (e.g.PCR) in which the template to be amplified comprises a complex mixtureof nucleic acid fragments representative of a whole (or substantiallywhole genome).

Solid-Phase Amplification

Once formed, the library of templates prepared according to the methodsdescribed above can be used for solid-phase nucleic acid amplification.

Thus, in further aspects the invention provides a method of solid-phasenucleic acid amplification of template polynucleotide molecules whichcomprises:

preparing a library of template polynucleotide molecules which havecommon sequences at their 5′ and 3′ ends using a method according to thefirst aspect of the invention described herein and carrying out asolid-phase nucleic acid amplification reaction wherein said templatepolynucleotide molecules are amplified.

The term “solid-phase amplification” as used herein refers to anynucleic acid amplification reaction carried out on or in associationwith a solid support such that all or a portion of the amplifiedproducts are immobilised on the solid support as they are formed. Inparticular, the term encompasses solid-phase polymerase chain reaction(solid-phase PCR), which is a reaction analogous to standard solutionphase PCR, except that one or both of the forward and reverseamplification primers is/are immobilised on the solid support.

Although the invention encompasses “solid-phase” amplification methodsin which only one amplification primer is immobilised (the other primerusually being present in free solution), it is preferred for the solidsupport to be provided with both the forward and the reverse primersimmobilised. In practice, there will be a “plurality” of identicalforward primers and/or a “plurality” of identical reverse primersimmobilised on the solid support, since the PCR process requires anexcess of primers to sustain amplification. References herein to forwardand reverse primers are to be interpreted accordingly as encompassing a“plurality” of such primers unless the context indicates otherwise.

As will be appreciated by the skilled reader, any given PCR reactionrequires at least one type of forward primer and at least one type ofreverse primer specific for the template to be amplified. However, incertain embodiments the forward and reverse primers may comprisetemplate-specific portions of identical sequence, and may have entirelyidentical nucleotide sequence and structure (including anynon-nucleotide modifications). In other words, it is possible to carryout solid-phase amplification using only one type of primer, and suchsingle-primer methods are encompassed within the scope of the invention.Other embodiments may use forward and reverse primers which containidentical template-specific sequences but which differ in some otherstructural feature. For example one type of primer may contain anon-nucleotide modification which is not present in the other.

In other embodiments of the invention the forward and reverse primersmay contain template-specific portions of different sequence.

In all embodiments of the invention, amplification primers forsolid-phase PCR are preferably immobilised by covalent attachment to thesolid support at or near the 5′ end of the primer, leaving thetemplate-specific portion of the primer free for annealing to it'scognate template and the 3′ hydroxyl group free for primer extension.Any suitable covalent attachment means known in the art may be used forthis purpose. The chosen attachment chemistry will depend on the natureof the solid support, and any derivatisation or functionalisationapplied to it. The primer itself may include a moiety, which may be anon-nucleotide chemical modification, to facilitate attachment. In oneparticularly preferred embodiment the primer may include asulphur-containing nucleophile, such as phosphorothioate orthiophosphate, at the 5′ end. In the case of solid-supportedpolyacrylamide hydrogels (as described below), this nucleophile willbind to a “C” group present in the hydrogel. The most preferred means ofattaching primers and templates to a solid support is via 5′phosphorothioate attachment to a hydrogel comprised of polymerisedacrylamide and N-(5-bromoacetamidylpentyl) acrylamide (BRAPA).

It is preferred to use the library of templates prepared according tothe first aspect of the invention to prepare clustered arrays of nucleicacid colonies, analogous to those described in WO 00/18957 and WO98/44151, by solid-phase PCR amplification. The terms “cluster” and“colony” are used interchangeably herein to refer to a discrete site ona solid support comprised of a plurality of identical immobilisednucleic acid strands and a plurality of identical immobilisedcomplementary nucleic acid strands. The term “clustered array” refers toan array formed from such clusters or colonies. In this context the term“array” is not to be understood as requiring an ordered arrangement ofclusters.

Use in Sequencing/Methods of Sequencing

The invention also encompasses methods of sequencing amplified nucleicacids generated by whole genome or solid-phase amplification. Thus, theinvention provides a method of nucleic acid sequencing comprisingamplifying a library of nucleic acid templates using whole genome orsolid-phase amplification as described above and carrying out a nucleicacid sequencing reaction to determine the sequence of the whole or apart of at least one amplified nucleic acid strand produced in the wholegenome or solid-phase amplification reaction.

Sequencing can be carried out using any suitable“sequencing-by-synthesis” technique, wherein nucleotides are addedsuccessively to a free 3′ hydroxyl group, resulting in synthesis of apolynucleotide chain in the 5′ to 3′ direction. The nature of thenucleotide added is preferably determined after each nucleotideaddition.

The initiation point for the sequencing reaction may be provided byannealing of a sequencing primer to a product of the whole genome orsolid-phase amplification reaction. In this connection, one or both ofthe adapters added during formation of the template library may includea nucleotide sequence which permits annealing of a sequencing primer toamplified products derived by whole genome or solid-phase amplificationof the template library.

The products of solid-phase amplification reactions wherein both forwardand reverse amplification primers are covalently immobilised on thesolid surface are so-called “bridged” structures formed by annealing ofpairs of immobilised polynucleotide strands and immobilisedcomplementary strands, both strands being attached to the solid supportat the 5′ end. Arrays comprised of such bridged structures provideinefficient templates for nucleic acid sequencing, since hybridisationof a conventional sequencing primer to one of the immobilised strands isnot favoured compared to annealing of this strand to its immobilisedcomplementary strand under standard conditions for hybridisation.

In order to provide more suitable templates for nucleic acid sequencingit is preferred to remove substantially all or at least a portion of oneof the immobilised strands in the “bridged” structure in order togenerate a template which is at least partially single-stranded. Theportion of the template which is single-stranded will thus be availablefor hybridisation to a sequencing primer. The process of removing all ora portion of one immobilised strand in a “bridged” double-strandednucleic acid structure may be referred to herein as “linearisation”.

Bridged template structures may be linearised by cleavage of one or bothstrands with a restriction endonuclease or by cleavage of one strandwith a nicking endonuclease. Other methods of cleavage can be used as analternative to restriction enzymes or nicking enzymes, including interalia chemical cleavage (e.g. cleavage of a diol linkage with periodate),cleavage of abasic sites by cleavage with endonuclease, or by exposureto heat or alkali, cleavage of ribonucleotides incorporated intoamplification products otherwise comprised of deoxyribonucleotides,photochemical cleavage or cleavage of a peptide linker.

It will be appreciated that a linearization step may not be essential ifthe solid-phase amplification reaction is performed with only one primercovalently immobilised and the other in free solution.

In order to generate a linearised template suitable for sequencing it isnecessary to remove “unequal” amounts of the complementary strands inthe bridged structure formed by amplification so as to leave behind alinearised template for sequencing which is fully or partially singlestranded. Most preferably one strand of the bridged structure issubstantially or completely removed.

Following the cleavage step, regardless of the method used for cleavage,the product of the cleavage reaction may be subjected to denaturingconditions in order to remove the portion(s) of the cleaved strand(s)that are not attached to the solid support. Suitable denaturingconditions will be apparent to the skilled reader with reference tostandard molecular biology protocols (Sambrook et al., 2001, MolecularCloning, A Laboratory Manual, 3rd Ed, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor Laboratory Press, NY; Current Protocols, edsAusubel et al.).

Denaturation (and subsequent re-annealing of the cleaved strands)results in the production of a sequencing template which is partially orsubstantially single-stranded. A sequencing reaction may then beinitiated by hybridisation of a sequencing primer to the single-strandedportion of the template.

Thus, the nucleic acid sequencing reaction may comprise hybridising asequencing primer to a single-stranded region of a linearisedamplification product, sequentially incorporating one or morenucleotides into a polynucleotide strand complementary to the region ofamplified template strand to be sequenced, identifying the base presentin one or more of the incorporated nucleotide(s) and thereby determiningthe sequence of a region of the template strand.

One preferred sequencing method which can be used in accordance with theinvention relies on the use of modified nucleotides that can act aschain terminators. Once the modified nucleotide has been incorporatedinto the growing polynucleotide chain complementary to the region of thetemplate being sequenced there is no free 3′-OH group available todirect further sequence extension and therefore the polymerase can notadd further nucleotides. Once the nature of the base incorporated intothe growing chain has been determined, the 3′ block may be removed toallow addition of the next successive nucleotide. By ordering theproducts derived using these modified nucleotides it is possible todeduce the DNA sequence of the DNA template. Such reactions can be donein a single experiment if each of the modified nucleotides has attacheda different label, known to correspond to the particular base, tofacilitate discrimination between the bases added at each incorporationstep. Alternatively, a separate reaction may be carried out containingeach of the modified nucleotides separately.

The modified nucleotides may carry a label to facilitate theirdetection. Preferably this is a fluorescent label. Each nucleotide typemay carry a different fluorescent label. However the detectable labelneed not be a fluorescent label. Any label can be used which allows thedetection of an incorporated nucleotide.

One method for detecting fluorescently labelled nucleotides comprisesusing laser light of a wavelength specific for the labelled nucleotides,or the use of other suitable sources of illumination. The fluorescencefrom the label on the nucleotide may be detected by a CCD camera orother suitable detection means.

The invention is not intended to be limited to use of the sequencingmethod outlined above, as essentially any sequencing methodology whichrelies on successive incorporation of nucleotides into a polynucleotidechain can be used. Suitable alternative techniques include, for example,Pyrosequencing™, FISSEQ (fluorescent in situ sequencing), MPSS(massively parallel signature sequencing) and sequencing byligation-based methods.

The target polynucleotide to be sequenced using the method of theinvention may be any polynucleotide that it is desired to sequence.Using the template library preparation method described in detail hereinit is possible to prepare template libraries starting from essentiallyany double or single-stranded target polynucleotide of known, unknown orpartially known sequence. With the use of clustered arrays prepared bysolid-phase amplification it is possible to sequence multiple targets ofthe same or different sequence in parallel.

Kits

The invention also relates to kits for use in preparing libraries oftemplate polynucleotides using the method of the first aspect of theinvention.

Preferred embodiments of the kit comprise at least a supply of amismatched adapter as defined herein, plus a supply of at least oneamplification primer which is capable of annealing to the mismatchedadapter and priming synthesis of an extension product, which extensionproduct would include any target sequence ligated to the adapter whenthe adapter is in use.

The preferred features of the “mismatch” adapters for inclusion in thekit are as described elsewhere herein in relation to other aspects ofthe invention. The structure and properties of amplification primerswill be well known to those skilled in the art. Suitable primers ofappropriate nucleotide sequence for use with the adapters included inthe kit can be readily prepared using standard automated nucleic acidsynthesis equipment and reagents in routine use in the art. The kit mayinclude as supply of one single type of primer or separate supplies (oreven a mixture) of two different primers, for example a pair of PCRprimers suitable for PCR amplification of templates modified with themismatched adapter in solution phase and/or on a suitable solid support(i.e. solid-phase PCR).

In one embodiment the kit may include supplies of different primer-pairsfor use in solution phase and solid phase PCR. In this context the“different” primer-pairs may be of substantially identical nucleotidesequence but differ with respect to some other feature or modification,such as for example surface-capture moieties, etc. In other embodimentsthe kit may include a supply of primers for use in an initial primerextension reaction and a different primer-pair (or pairs) for solutionand/or solid phase PCR amplification.

Adapters and/or primers may be supplied in the kits ready for use, ormore preferably as concentrates requiring dilution before use, or evenin a lyophilised or dried form requiring reconstitution prior to use. Ifrequired, the kits may further include a supply of a suitable diluentfor dilution or reconstitution of the primers. Optionally, the kits mayfurther comprise supplies of reagents, buffers, enzymes, dNTPs etc foruse in carrying out PCR amplification. Suitable (but non-limiting)examples of such reagents are as described in the Materials and Methodssections of the accompanying Examples. Further components which mayoptionally be supplied in the kit include “universal” sequencing primerssuitable for sequencing templates prepared using the mismatched adaptersand primers,

The invention will be further understood with reference to the followingnon-limiting experimental example:

EXAMPLE Experimental Overview

The following experimental details describe the complete exposition ofone embodiment of the invention as described above. The DNA source usedis purified Human cell line DNA supplied by the Coriell CellRepositories, Camden, N.J. 08103 USA, catalog no. NA07055. The DNA isfirst prepared for ligation to forked adapters by: fragmentation of theDNA by nebulisation, end repair of the DNA ends to make them blunt-endedand phosphorylation, then the addition of a single ‘A’ nucleotide ontothe 3′ ends of the human DNA fragments. The ligation reaction isperformed with the prepared fragmented DNA and adapters pre-formed byannealing ‘Oligo A’ and ‘Oligo B’ (sequences given below). The productof the reaction is isolated/purified from unligated adapter by gelelectrophoresis. Finally, the product of the ligation reaction issubject to cycles of PCR to selectively amplify ligated product thatcontains adapter at both ends of the fragments.

Materials and Methods Nebulization Materials:

-   -   Human genomic DNA (1 mg/ml) Coriell NA07055    -   Buffer (glycerol 53.1 ml, water 42.1 ml, 1 M TrisHCl pH7.5 3.7        ml, 0.5 M EDTA 1.1 ml)    -   Nebulizer Invitrogen (#K₇₀₂₅-05)    -   Qiagen columns PCR purification kit (#28104)        Mix: 25 μl (5 micrograms) of DNA    -   725 μl Buffer

Procedure:

Chilled DNA solution was fragmented in the nebulizer on ice for 5 to 6minutes under at least 32 psi of pressure. The recovered volume (usuallysomewhere between 400 and 600 μl) was split into 3 aliquots and purifiedwith a Qiagen PCR-purification kit, but using only one column, andfinally eluted in 30 μl of EB (Qiagen).

End-Repair Materials:

T4 DNA Polymerase NEB #M0203S 10xNEB 2 buffer NEB #M7002S 100x BSA NEB#M9001S dNTPs mix (10 mM each) NEB #N0447S E. coli DNA Pol I largefragment (Klenow, NEB #M0210S) T4 polynucleotide kinase NEB #M0201S T4PNK buffer NEB #M0201S 100 mM ATP Qiagen columns PCR purification kit(#28104)End repair mix was assembled as follows:

DNA 30 μl Water 12 μl 10xNEB2 5 μl 100xBSA 0.5 μl 10 mM dNTPs 2 μl T4DNA pol (3 U/μl) 5 μl 50 μl totalThe reaction was incubated for 15 min at room temperature, then 1 μl ofE. coli DNA Pol I large fragment (Klenow) added and the reactionincubated for a further 15 min at room temperature. The DNA was purifiedfrom enzymes, buffer, etc by loading the reaction mix on a Qiagencolumn, finally eluting in 30 μl EB. The 5′ ends of the DNA were thenphosphorylated using polynucleotide kinase as follows:

DNA 30 μl Water 9.5 μl 10xPNK buffer 5 μl 100 mM ATP 0.5 μl T4 PNK (10U/μl) 5 μl 50 μl totalThe reaction was incubated for 30 min at 37° C., then heat inactivatedat 65° C. for 20 min. DNA was then purified from enzymes, buffer, etc byloading the reaction mix on a Qiagen column, finally eluting in 30 μlEB. Three separate tubes were pooled to give 90 μl total.

A—Tailing Reaction Materials:

Taq DNA polymerase NEB #M0267L 10x thermopol buffer NEB #B9004S 1 mMdATP Amersham-Pharmacia #272050 Qiagen MinElute column PCR purificationkit (#28004)The following reaction mix was assembled:

DNA 30 μl 10x thermopol buffer 5 μl 1 mM dATP 10 μl Taq pol (5 U/μl) 3μl ~50 μl totalThe reaction was incubated for 30 min at 70° C., then the DNA purifiedfrom enzymes, buffer, etc by loading the reaction mix on a QiagenMinElute column, finally eluting in 10 μl EB.

Anneal Forked Adapter Materials:

-   -   ‘Oligo A’ and ‘Oligo B’    -   50 mM Tris/50 mM NaCl pH7    -   PCR machine

100 μM Oligo A 20 μl 100 μm Oligo B 20 μl Tris/NaCl 10 μl 50 μl at 40 μMduplex in 10 mM Tris/ 10 mM NaCl pH 7.5 Oligo A:5′ACACTCTTTCCCTACACGACGCTCTTCCGATCxT (x = phosphorothioate bond) OligoB: 5′Phosphate-GATCGGAAGAGCTCGTATGCCGTC TTCTGCTTGThe adapter strands were annealed in a PCR machine programmed asfollows:

Ramp at 0.5° C./sec to 97.5° C. Hold at 97.5° C. for 150 sec

Then a step of 97.5° C. for 2 sec with a temperature drop of 0.1°C./cycle for 775 cycles

Ligation Reaction Materials:

40 μM forked adapter A-tailed genomic DNA Quick Ligase NEB #M2200L QuickLigase 2x buffer NEB #M2200L PCR machine Qiagen columns PCR purificationkit (#28104)Reaction mix was assembled as follows:

DNA 10 μl 2× buffer 25 μl 40 μM adapter 10 μl Quick Ligase 5 μl ~50 μltotalThe reaction was incubated for 20 min at room temperature then the DNApurified from enzymes, buffer, etc by loading the reaction mix on aQiagen column, finally eluting in 30 μl EB.

Gel Purification Materials:

Agarose Biorad #161-3101 100 base pair ladder NEB #N3231L TAE Loadingbuffer (50 mM Tris pH8, 40 mM EDTA, 40% w/v sucrose) Ethidium bromideGel trays and tank. Electrophoresis unitThe entire sample from the purified ligation reaction was loaded intoone lane of a 2% agarose gel containing ethidium bromide and run at 120Vfor 50 min. The gel was then viewed on a ‘White-light’ box and fragmentsfrom above 300 bp to at least 750 bp excised and purified with a QiagenGel purification kit, eluting in 30 μl EB. For large gel slices twominElute columns were used, eluting each in 15 μl EB and pool.

PCR Amplification Materials:

Ligated DNA PRIMER 1: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCT ACACGAPRIMER 2: CAAGCAGAAGACGGCATACGA 2x Jump Start RedTaq PCR mix Sigma#P0982 PCR machine Qiagen MinElute columns Qiagen(#28004)The purified ligated DNA was diluted 25 fold, then a PCR reaction mixprepared as follows:

DNA 1 μl 2x Jump Start RedTaq mix 25 μl 100 μM Primer 1 0.5 μl 100 μMPrimer 2 0.5 μl Water 23 μl ~50 μl totalThermocycling was carried out in a PCR machine under the followingconditions:

2 min @ 94° C.

-   -   [45 sec@ 94° C., 45 sec @ 65° C., 2 min @ 70° C.] 16 cycles    -   5 min @ 70° C.    -   Hold @ 4° C.        PCR products were purified from enzymes, buffer, etc on a Qiagen        MinElute column, eluting in 10 μl EB. The resulting DNA library        is ready for amplification on a surface PCR platform.

Validation of Library

1 μl of the DNA library was cloned into a plasmid vector and plated outon agar. 19 colonies were picked, miniprepped and the cloned insertssequenced by conventional Sanger sequencing. The sequence data obtainedwas as follows:

Clone 1 TGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGTGGGGACCGTCCTGTGCATTGTAGGGTGTTCAACAGCATCCCTGACCTCCACCTACAAGATGCCAGTAGCGAATCCCCTCAGCCCTCATCTCCTTGCCATAGTTGTGTCAACCAAAATCATCTCCACACATTGTTAGATGTTTACTGGGAGGCAGACTCACTCCCACTTGAGAACCACTGTACTAGAAATATCACCAAGAGAATGAGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG Clone 2AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGTGGGCTTTGTTCTTTGAGAGGTTGCAGTCAACATGATTCTTTAAGACCAGAACCCTGCACACTTCTTGGGCTGTATTTCTTACATTCCTTTTCTATTTTAACCATATCCCATCTTACCTACTTCCAGCATAGTGGTCATATTTAATTTTTACAAAACCATTTTGCCACTTGCTGCCAACTATGTTCTTTATAAAGCAGACTTTGAGATGGAGGCTAGTGTTCAGAGGGGATGCTTAGGAGAACTTTGGAGATTAATACTTATGGCAGGTAAGGGAAGGAAGCAGGATTAGACAGAAATATTGAACTGTGATACAAAGTCAGCAAAGACTTTAGTCAATAGATCGGAAGAGCTCGTATGCCG TCTTCTGCTTGClone 3 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTTCGATTCCCTTCAATGATTATTCCATTCGAGTACATTCGATGATTCCATTCGATTCTATATGATGATGATTGCATTCGAGTCCGTGTATTATTCCATTCCATTCCATTAGATGATTCCATTCGAGTCCATTCGATGATTCTCTTCGATTCCGTTCGATAATTACGCTTGATTCCGTTTGATGTTGATTCCATTCGAGTCCATTCAATGTTAATTCCATTCGATTCTAAGCGATGATTCCATTCCTTTCCATTAGAAGATGATTCCATTCGAGACCATTCGATGATTGCATTCAACTCATTCGATGACGATTCCATTCAATTCTGTTCAATGATTCCATTAGATTCCATTTGATGATGAGTCCATTCGATTCCATTTGATGATGATTCCATGCGATTCCATTAGATGATGACCCCTTTCATTTCCAAGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG Clone 4AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTTAAATGCTAGGCATATTGTGTACCCCACATTGGTTTGTAGCCAGCTCTATGTCATAGGGCCCTTACCCTTTACCTATTTATTGTTAGTATAATGTCCATAAACAAGCCAATGGCTCAGCATGAACTGATGCTAAAGAAAGCTCATGCCTGAGTGATAAATTAAGTGACCTCAGCTATTTCTCTTCAGTGTTGTGAAAGTTATTTTTAACAGTAGGTTTCCTGGTAGATTCTCTAACCACTCGGTATTTCACATGGCCCAACTTGGTTAACTCGACTGGTTACGGCAAATGCTGAAGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG Clone 5AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAAGCTCTTCCGATCTTAAGGAAGTTAGGTAGATAATTTTTGTTTAGGCCATATAGCTTTGATTTTCTGATAACAATTTTATAAACTTAGAAATTTTCATGTAAGATACAGGAATACTGGAAGCAAAAAAAAGAAGGTGCTTTAACCTTAGGGATTGAAAAAATAGTAATTTAGGTTGAAAATGCTGCTTGAAAGTTAATGCTGATAGCATTACTACACATGATGATTTTTTCTGGAAGGAAAGCTTTATCTGGGCCTTCAATTTAGGAATTTTTCTCTTTGGTTTTTAAAAGCTGCCATATTCACTTGAGCTTCATGGGAAAGATGCAAATAACTAAAACAAATGAACAAAAACCATGTTGAGGTCAGGAACTTATTTCAAGAAAGCAAGTTCTAGGTTTTCTTTTAAAGTGACAGTAGAGCCTTAGGCCTCAAACCATCTACAACCATGTTAACAGTAAGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG Clone 6AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCAGATCTTCCTGCCTCAGCCTCCCGAGTAGTTGGGATTGCAGGCATGTGCCACCATGCCCTGCTAATTTTTGTATTTTTAACTAAAGGAGGGGTTTTGCCATGTTGGCCAGGCTGGTCTTGAATGCCTGACCTCAGGTGATCCGCCCACCTCAGCCTCCCAGTGCTGGGATTACAGGTGTGAGCCACTGCGCCCAGCTGAGGGTAACTATTTTTAATGTGGCTGATGAATGTAACTATCCTGTCCCATGTCTCTGTCCCCAGCTGCAGAGCCCTCGTCGAGATCGGAAGAGCTCGTATGCCGTCTTCTG CTTG Clone7 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTAAGGTTTAAATTTTCTATATAGCCTGAAAAGTTTGAATGTTTAATTCAAACTAATTTATTGAGCAATGGCATTTAAGAAAATGGAAAGATACAAAGGGACTTTCATCAGATGATAAGTGGATAAGAGAGAAAAATGCAGACAGATGAGCCAGAGTTGTGTAAAAGCTGGGAGGCTAGCAGGGCCTTGTAGATAGCCAAGCTGACTGGGGAACAGAGATAAATGGGAGCAGAGATCAGAAAGTTCATCCTTACCCTGCTGCCCGTGGTGAAAGGAGACTTGCAAGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG Clone 8AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTAAGGAATCTTTATTTTCTACATTTGAGTTTGGAAAACTGAGCTAGCACATCTAAATCCATCTAATTTTGGTCATTGGTTTTAACAAGTTCATCTTATTTTTTTAAACATCTGATCTTTATTTTATAGAATAGACTACACAAAGTCTTTTGGAAAATTAAAATATTTTAACTTCCAACAATTTTCAGATTTTACTTATAAAAAAATTTAAAATCCTCTACTTTACTCGCATCTTTATTATTTCTGACTTTCTAGCTACTTAAAGTTAAGGAGGAAATTAACCTCTCTAAGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG Clone 9AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTCGCCATAACCACAGCCCAGGCCTCCGTAGCCACAGCACCCATAGCCATGGCCACCATTGTAGTTTCTGTAGTAGCTGCCACACATGGTGTTGGTTGTTGAGGTCATCCTTGGGTAGGAAGGAGTGTAGGTGACTTCAGTATGGACACTTCTCCGCAGAGGGCCTTTTATATGCCTCAGTGAATCAGAACATAGCGTGCCCCTGCAAAAATATCTCTAAAGGCCTTTCATTGTGCTGAGAAGTTCTGGCCCTTACGTATCTCTCTGATTTCATATCCTGCTACTCTCCTCCATTTATCTATAATGCTCAAACTCTGCTGGCTTTTTGTCTTTTAAAATGCAGCAGGTTTCTTCTCACAATAAGGAGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG Clone 10AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTAGAGAGAAGTTATTTAAGACAAGTAAGGTATCAGGTTGTGACTCAAATACCACAGAATCTAGCTATTGTTAGCAATATTAAGTATATTTTCTTAAATGAAGGATTCTCCATTTACCATATGCCCCTTGGATAATTTCCAGAGAATTTAATTTTTTAAAAGGAATTTTCACCAATTAAATTATTGTTTTGATCAAAAGAGGACCCACTGAACACCTTATTCATTATTAAAATGTATCATAAAACTTAATTATGGAGCTGGGTACAGGGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCTGAGGCAGGAGGACTGCTTGAGTCCAGGAGTTTGAGACCAGCCTGGGTAACATGGTGAAACCCTGTCTCTACAAAAAATACAAAAAATTAGCCAGGTGTGGTGAGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG Clone 11AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTCCCAGGGAAGCCAAAAGATTGGACACCCCTCTTTCAAACTATAAATTCCTCCCATAGTTAGTTTGGCCTATGCTGGGAAATGAACAAGGGTGGCTTTGAGGTTAGAAGCAAAATGGAGTCAGTTAGGTCAGACTTTTTTTCACTATCATACTTTTTCTATGTCAGATTTATCTCACTTGTAATTTTTGCAAGGGTGGTTTCAGAGCCACTAAGCTTGTGGTAATTTTTTACTGCAATAGAAAACTAATGCATTAGGTAACCCTCTTTTTTTCCCTCTGATTGCTTGCTCTGGGGTAAGCCAGCTGCCATGTTGAATTTTTCATTTTGTTACTGAGCAATCAATGGGCTCACTGCCCGACGTGCATAGAGGCCAATACTGTGGCACTAGTTTTTGAGAAAAGATCGGAAGAGCTCGTATGCCG TCTTCTGCTTGClone 12 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTTTCAGATTTTATATGTATTAACTCAGAACACACACCTCTTATCACACATATTTTTTCATGTAATTTATCTAAATCTTATAGAAAAGGGTCCATTTGCATTTTCTCTTATTAGACTCCTGATTTCAAATAATATATTACTTATGAGTATTTTTCTGTGCTGTAGTTATTCATTCTTATAGATATGTAACATAATTCCTTTTGCAAAGGTAAAAATTGAGCTATCTCTTGTTGAGGATTTGTTGATCTCTGTCTAAAGTTTCAAAAATAAAGAACTTTAAAAGCAAAATGTAAATTCCTTTCAAGTTTTAGTAAAATTACTTCAAACTTAGTAGCTTAAACAATACAGATTTATTATGTTACAGTTCTGTAAGACAGAAATCTGACTTGATCACACCATGGTAAAACCAAGATACTGCCAGGGTTGGTTTTTTCTTGGGGGGGGTCTGTGGGAAGAGTTTGTTTCCTTTGGTTTTCCACAGCCCAGAGGCTGCTTGCATTCCTTTGATCACTAGATCGGAAGAGCTCGTATGCCGTCTTCTGC TTG Clone13 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTATTGTTTAGGGAATAATGACAAGGAAAAAAAGTCTGTAGATATTCAGTACAGAGGCACCCATCTTTTTAAATTTCTGAAGATTTTTTACTCATGCTTGGTTGAATCCACAGATGCAGAACCCATAGGTTCAGAGGGCCAGCTGTGCTTTGAAAATATTAGCTTGTGTTTTTATTAGAAAGAAAACTCTGAGGCCAGGCACGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCTGAGGTGGGCGGATCACAAGGTGAGGAGATCGAGACCATTCTGGCTAACATGGTGAAACCCTGTCTCTACTAAAAATACAAAAAAATTAGCCGGGCGTGGTAGTGAGCACCTAGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG Clone 14AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGCAATTGGTAAAACAGTAAGCAATGAAACAGACACTTCTCAAATATTCCAAGATGGTACACGCTTTTCAGTGTGTATGATCCAATAAAGCCATTGGAAGTAGGCTTTAATAGTCAAAAAAGACTATTCAGTTAGATAGGAACTATTTGCCTATAACTATTGGCCAAAAATAGGTTAAAAAATTGTTTTAAATTTGTGCTTTACAAAACATGTGGACTTTTTTAGAAAATGTGTCAAATTTCAAAAGAAATATAGACATTATGGAAAGGTCAGTTAAGCACAGCCCTAATCCTGAAAACATAACTATGAAAGATACTAGCTGTTACTTGTAACCAAAAGGAAAAAAAAGATATTAGTAACCAATAATTAGCAAACAATGCCCATATATTTCCTTTTTTTTTTTTTTTTTTTGAGACAGGGGCTTACTCAGGCTGGATGTGATCATGAGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG Clone 15AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTTAGAAGCTCTATTATACTGGAAAAGAGATATGAGACCCTTCCTACTTTAAGAATCAATGAAGCCGGGTGTGGTGGCTCACGCCTGTACTCCCAGCATTTTGAGAGGCCAAGCTGGGCAGATCACCTGAGGTCGGGAGTTTGAGACCAGCCTAGCCAACATGATGAAATCCTGTTTCTACTAATAACACAAAAATTAGCCGGGTGTGGTGGCGCACATCTGGAATCCCAGCTACTCCAGAGGCTGAGGCAGGAAAATTGCTTGAAGCTGGGAAGCAGAAGTTGCAGTGAAGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG Clone 16AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGGCTGGACTGAATAGGATAGCCTTAGCTGTAAAATTGGGCTGATCTTTCAAATGGACTCATGCTTGCCGAATGACTCACGCTCCTGTTTACAAATCAGCTCTGTGAAGAAATGCAGAGTGGGAGGCTCTGCTTGCCAGACGGAGACCTTAGACCTCCAGGGGCGGAGAACGGAGTACTTCCTCTGGTGCTCGGCTTCCCTTCCTGGGGGCAGATCTCTCAGCTTCTGGTTGGTGGCTCTCAAAATCCAGACACAAGGTCAGCTGCAGCCAGCGTGGGCCCTGGAGTAGCTCCAGTTATGGGGCAGCAATGGCCCCCTCTCATTTTGAGAGCTCACTTTGCCTGTGGATGGTTTTAATCCATCTGGATAAACTTGAGGCCCATGGGAATACCATATACTATGGTAACCATGTACACTGCTCTAAAGATGTGGCTGCTGTTGTATAACTTTTTCCTTTATTTTTGTCAATTTCCTATTTTCCAGAGTCTTGCATACCCACTATGTCTACTGTGATAGTGAACGTAAAAACATACAAGATGTTGGTGTTATCCTCAATCTCAGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG Clone 17AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTCTGTGAGAAATCAATGTCTGCTGTTTATAAGCCGCCGGGCTGTGATATCCTGTGAGAGTGGCCCCAGTGGATGAAGACAGATGCTCTCAAGGAGCGCAGATGACGCGGGTTCCGAAGGACTCGGCACCCAGCCCGGAGGCCGGCAACATGGGCAAGGGGCCTCTCACGGCTGACCTGTTTCCTCATCAGCACATCAGGACAATAAGAGCTCCCACTTCACAGGTGGTGAAGAGCCAACGTGGTGAAGAATGAATAAAGCAGCTCGTGGAAAGTGCTGTGCATGAGGCCTGGCAACCGGTCCCTGCTCTGAGGTCACCTGCCACGGAGCTGCTGACAGGACCATTAAAAACACAATTGTGCAAGTGCTCACCCACATTCACAGCAGCAGAATCTCCACCAGCCAAGCATTGGAGACGATCCTTGCATCCATAGACATGAACAGATGAGCAAAACGTGGTCTATACGGACGATGAAATAGCACTCAGCCCTAAGAAGAAATAAAATCCCGACAGAGAGATCGGAAGAGCTCGTATGCCGTCTTC TGCTT Clone18 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGCTGAGAGGAGAGGAGAGGCTGGCAGGGGGCTGATGCAGGAGGTGATAGGGCTCCCGGTGATAAGAGGTGAGAAGAACAGTCTCTGTGTGCCTAGAGAAGAGATTACCAGAAGTCTGCTATCTGTTTGTTCGCGGATGTCGGACAGGCAGGATCGGTGATGGCAGGTCTTGGGGGAAGGATTATCAGGAGCTAAAAGCTGTCTTCACCTTGGCTGCTAAGAACTCATCTCGGATCTTCTTAGAATTCCAAATCGGACTTTTCTCCTAGCAGTGGCTACATCCTTAACCTCAAAAATACCCGTATTAGCAGATCTACCTCCATGAAATAGACAATTCTTGACAAACTAAGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG Clone 19AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGTTATTTTTCATACTGATGATTCTAGGGCGGTTCCCTGGACTTGGCTGGGACCAGAACTGTCTCACAGCTGCCAGCGCTCCCCACCCCCATGAGAACCATCGTGCTGAAGATGAAGAGCCAAAGCCAAACTGACCAGCCTCTGCAACCCCAAGCATCACCGTGTGAGGACAGCGTGATGTGGAGCCGCGGAGACCCCACGCAGTGCTGCGAGGGGCAACCACATGCAGGGGGGCAGAGGTGTGGGGGAGCAAGCAGATGCCACCGGCATGCCTAGGAGCCACAGGGAGCATGCGGGCTGGGCAGTGATGGGAATGAACGTGAACTCCAGTCCTGCCCCAAGAAGTCCTCCGGCCCCTCCTTCTCAATTCAGGGCACAAAGTGGTAACTGCAGCATGCAGAGGAGTCGAGGAGTCTTCCCTCGTCCCAGGAGCAGCACCTCGGGCACAGTCTCGGTCCCACAGAACAGCCAAGTGTGGGTTGGTGGTCTAGAGACCTCCGAAGATCCAGTGGGGGAAGGATGGGCAGCAGAGGGTCTACTCTCTGAAAATAAGGGGAAGGGATTTTCCCTCCCCACTGCCAAGGTCCCAGCTACTGGACGTGGGTGGAGATCGGAAGAGCTCGTATGCCGTCTTCTGCCTTThese results confirm that the library preparation method produces alibrary of “sequenceable” DNA templates containing a mixture offragments of different sequence.The insert DNA from each of the 19 clones sequenced was found to alignto a human genome reference sequence (alignment not shown), illustratingthat the method produces a library of templates which truly reflect thesequence composition of the starting target fragments (i.e. the clonescontain human genomic fragments rather than “junk” sequence).

1-29. (canceled)
 30. A method of generating a library of different polynucleotide molecules, the method comprising: (a) providing a plurality of different target polynucleotide duplexes; (b) providing identical polynucleotide adapters, wherein each adapter comprises a double-stranded annealed region and a mismatched region; (c) ligating the double-stranded annealed regions of the identical polynucleotide adapters to both ends of the different target polynucleotide duplexes to form adapter-target constructs, and (d) annealing a single universal primer species to the mismatched regions of the adapter-target constructs and extending the primer to form extension products complementary to both strands of the adapter-target constructs, wherein the extension products comprise nucleic acid sequences that differ from the nucleic acid sequences of either strand of the adapter-target constructs and the extension products collectively provide a library of different template polynucleotide molecules which have common sequences at their 5′ ends and common sequences at their 3′ ends, wherein the common sequences at the 5′ ends of the extension products are not complementary to the common sequences at the 3′ ends of the extension products.
 31. The method of claim 30, wherein the annealing and extending in step (d) comprises: i) annealing a single universal primer species to the mismatched regions of each of the adapter-target constructs, ii) extending the primer species by sequential addition of nucleotides to form extension products complementary to at least one strand of each of the adapter-target constructs, and iii) subjecting the products obtained in step ii) to denaturing conditions, thereby separating the extension products from strands of the adapter-target constructs.
 32. The method of claim 30, wherein the polynucleotide adapters are forked adapters formed by annealing of partially complementary first and second polynucleotide strands.
 33. The method of claim 32, wherein a sequence of 5 or more consecutive nucleotides at 3′ end of the first strand of the forked adapters is complementary to a sequence of 5 or more consecutive nucleotides the 5′ end of the second strand of the forked adapters such that a double-stranded region of 5 or more consecutive base pairs is formed by annealing of the two strands.
 34. The method of claim 33, wherein a sequence of at least 10 consecutive nucleotides at the 5′ end of the first strand of the forked adapters and a sequence of at least 10 consecutive nucleotides at the 3′ end of the second strand of the forked adapters are not complementary such that an unmatched region of at least 10 consecutive nucleotides on each strand remains in single stranded form when the double-stranded region is annealed.
 35. The method of claim 30, wherein the different target polynucleotide duplexes are fragments of genomic DNA.
 36. The method of claim 30, wherein the different target polynucleotide duplexes are double stranded cDNAs.
 37. The method of claim 30, further comprising carrying out a solid-phase nucleic acid amplification reaction wherein said library of different template polynucleotide molecules is amplified on a solid-phase surface.
 38. The method of claim 30, further comprising carrying out a solid-phase nucleic acid amplification reaction, wherein the library of different template polynucleotide molecules is amplified by solid-phase PCR using forward and reverse amplification primers and solid-phase PCR is carried out using primers identical to those used in preparing the library of different template polynucleotide molecules.
 39. The method of claim 30, wherein the single universal primer species comprises a non-hybridizing tail sequence at the 5′ end whereby the single primer species is extended to generate extension products that comprise the tail sequence or a complementary sequence thereof.
 40. The method of claim 39, further comprising carrying out a solid-phase nucleic acid amplification reaction wherein the library of different template polynucleotide molecules is amplified, wherein the library of different template polynucleotide molecules is amplified by solid-phase PCR using forward and reverse amplification primers and solid-phase PCR is carried out using amplification primers capable of annealing to the tail sequence of the extension products or the complementary sequence thereof.
 41. The method of claim 30, further comprising determining the sequence of at least a part of the different target polynucleotide duplexes in the library of different template polynucleotide molecules.
 42. The method of claim 30, further comprising a step of purifying the extension products from the adapter-target constructs.
 43. The method of claim 30, wherein the providing a plurality of different target polynucleotide duplexes comprises fragmenting a complex polynucleotide sample.
 44. The method of claim 43, wherein the library of different template polynucleotide molecules comprises sequences of the whole complex polynucleotide sample.
 45. The method of claim 44, wherein the complex polynucleotide sample comprises genomic DNA.
 46. The method of claim 44, wherein the complex polynucleotide sample comprises double stranded cDNAs.
 47. The method of claim 37, wherein amplification is carried out using a single surface bound primer.
 48. The method of claim 37, further comprising carrying out a sequencing reaction to determine the sequence of at least a part of at least one amplified polynucleotide molecule.
 49. The method of claim 30, wherein the library of different polynucleotide molecules is used to prepare a clustered array of nucleic acid colonies on a solid support.
 50. The method of claim 49, further comprising carrying out a sequencing reaction to determine the sequence of the nucleic acid colonies.
 51. The method of claim 30, further comprising amplifying the library of different polynucleotide molecules on a solid support.
 52. The method of claim 37, wherein the solid-phase surface is a solid support.
 53. The method of claim 52, wherein the method is carried out on an apparatus comprising the solid support.
 54. The method of claim 53, wherein the apparatus comprises a source of reactants and detector means for detecting one or more signals produced after the reactants have been applied to the amplified library of different template polynucleotide molecules.
 55. The method of claim 49, wherein the method is carried out on an apparatus comprising the solid support.
 56. The method of claim 55, wherein the apparatus comprises a detecting means for detecting and distinguishing signals from individual nucleic acid colonies arrayed on the solid support.
 57. The method of claim 52, wherein the solid support is selected from the group consisting of latex beads, dextran beads, polystyrene, polypropylene surface, polyacrylamide gel, gold surfaces, glass surfaces and silicon wafers.
 58. the method of claim 57, wherein the solid support is a glass surface. 